Research               Publications               Lab Members               Positions Available


Research


Our laboratory studies molecular and cellular mechanisms of vision. Most of our work is centered on the vertebrate photoreceptor cell, a sensory neuron responsible for the light detection in the eye. At this time, we are pursuing the following experimental directions.



 I. REGULATION OF LIGHT SIGNALING

The basic functions of photoreceptors are to capture photons, to generate a second messenger signal, to translate this signal into a change in electrical activity and, finally, to transmit this information to the secondary neurons in the retina through modulation of synaptic release. Because the function of this cell is so well-defined and because it is uniquely suitable for study using modern multi-disciplinary approaches, the photoreceptor is an almost unmatched model system for elucidating fundamental issues in molecular neuroscience and cell signaling. The most mature direction of our laboratory addresses how each of these steps in information flow, from photon capture to synaptic release, is regulated on the molecular level. We are particularly interested in learning how signal amplification, response duration and light adaptation are performed by the visual signal transduction cascade illustrated in this figure.


Schematic of the phototransduction cascade activation and deactivation


II. HOW TO BUILD A LIGHT-SENSITIVE ORGANELLE?

Photoreceptors are highly compartmentalized neurons, with all molecular events responsible for generating light-signals confined to the outer segment, a ciliary organelle containing a stack of flattened membrane vesicles (“photoreceptor discs”) enclosed within a plasma membrane. Building this organelle involves two interconnected processes – formation of its unique anatomical structure and populating it with a unique set of signaling and structural proteins. We are studying both of these processes. Our first goal is to reveal the intracellular trafficking pathways responsible for specific protein delivery to the outer segment. Almost everything we know today about outer segment protein trafficking relates to that of the visual pigment, rhodopsin. The mechanisms responsible for delivery of other outer segment-resident proteins remain elusive. It is not even clear whether delivery of other proteins utilizes the components of rhodopsin trafficking pathway, or several unique pathways coexist in photoreceptors. These conceptually distinct options are explored in our ongoing experiments. Our second goal is to elucidate the complex process of photoreceptor disc morphogenesis, which starts with evagination of the plasma membrane at the outer segment base, followed by lateral membrane outgrowth, flattening, and, in the cases of rods and mammalian cones, subsequent disc enclosure. We are aiming to identify proteins responsible for performing each of these specific tasks and to understand how they function as highly coordinated molecular ensemble.





An electron micrograph of the mouse rod outer segment and the connecting cilium bridging the outer segment with the rest of the cell. The newly forming discs are “open” and have higher electron density that the mature, enclosed discs.

A frog rod photoreceptors expressing fluorescent plasma membrane marker
shown in green. The outer segment is stained in red and nucleus in blue.

  

III. PHOTORECEPTOR PATHOBIOLOGY

Our studies of photoreceptor cell biology go hand-in-hand with understanding pathobiological processes underlying degenerative diseases of the retina, which lead to blindness in animals and human patients. Currently we pursue two directions. The first explores the novel concept that a major cellular stress factor contributing to photoreceptor cell death in multiple forms of retinal degenerative disorders is proteasomal overload, i.e. insufficient capacity of the ubiquitin-proteasome system to process abnormally large quantities of misfolded and/or mistrafficked proteins associated with these conditions. The second addresses the functional role of PRCD (Progressive Rod-Cone Degeneration), a small protein whose mutations serve as a leading cause of blindness in dogs and are also identified in human patients undergoing progressive visual loss. Our recent proteomic study identified PRCD as a constitutive component of photoreceptor discs. Our current experiments aim to pinpoint its specific role in these membranes and to understand why PRCD mutations cause photoreceptor degeneration and blindness.


Intracellular accumulation of the fluorescent reporter protein, UbG76V-GFP (green), associated with proteasomal overload in
photoreceptor cells degenerating due to various mutations. Outer segments are highlighted by a red lectin marker.



 IV. MASS SPECTROMETRY

One of the most powerful approaches to understanding the function of a cellular organelle is to know its protein composition. This is certainly true for photoreceptors, and we are engaged in determining protein compositions of their subcellular compartments using high-end mass spectrometry. Our favorite experimental strategy is protein correlation profiling, a powerful approach to analyze multi-protein complexes or organelles that can be fractionated but not purified to homogeneity. We recently published the unique proteome of photoreceptor discs. Our ongoing and planned projects include identification of protein compositions of the plasma membrane encapsulating the outer segment, the connecting cilium bridging the outer segment with the photoreceptor cell body, and transport vesicles carrying building materials from biosynthetic membranes to the outer segment.



Eleven transmembrane proteins uniquely residing in photoreceptor disc membranes.